MHC Class I Associated Hepatitis B Peptides

ABSTRACT

The present invention relates to compositions and methods for the prevention, treatment, and diagnosis of Hepatitis B virus (HBV) infection, and discloses peptides, polypeptides, and polynucleotides that can be used to stimulate a CTL response against HBV infection. The peptide and/or proteins of the invention may be used as a therapeutic drug to stimulate the immune system to recognize and eliminate HBV infection in infected cells or as a vaccine for the prevention of disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/168,324, filed on 23 Oct. 2018, which is a continuation of U.S.national application Ser. No. 15/121,496, filed on 25 Aug. 2016 now U.S.patent Ser. No. 10/155,036, which is the US national phase applicationof PCT/US2015/015807, filed on 13 Feb. 2015 now expired, which claimspriority to U.S. Provisional application No. 61/946,183, filed on 28Feb. 2014 now expired, the disclosures of which are herein incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of immunogens whosestructures incorporate peptides derived from HBV infection and tomethods of using such peptide as a basis for the prevention andtreatment of diseases such as HBV infection.

BACKGROUND OF THE INVENTION

The mammalian immune system has evolved a variety of mechanisms toprotect the host from microorganisms, an important component of thisresponse being mediated by cells referred to as T cells and byantibodies derived from B cells. In combating bacterial infections,antibodies are especially important but likewise are specialized T cellsthat function primarily by recognizing and killing infected cells. Thelatter also function by secreting soluble molecules called cytokinesthat mediate a variety of functions of the immune system. Thus, theimmune system is highly developed to deal with infectious organisms aswell as with the elimination of cells infected with such organisms.Among the latter are viral infections, such as HBV infection.

Hepatitis B virus (HBV) is a member of the Hepadnaviridae family ofviruses which also includes woodchuck hepatitis virus (WHV) and duckhepatitis B virus. These viruses are primarily hepatotropic withinfections characterized by fever, fatigue, muscle aches, and yellowingof the eyes and/or skin. The severity of these symptoms can vary with aproportion of cases being asymptomatic. More than 2.5 billion peopleworldwide have been infected by HBV, but for the vast majority of adultsencountering the virus (>90%), the infection is acute and readilycleared by the immune system. For the remaining 5-10% of adults, and forneonates and unvaccinated children, HBV establishes a chronic infection.Approximately 370 million people worldwide are chronically infected andover 500,000 people die each year due to complications from HBV. Thesecomplications include liver cirrhosis, liver failure, and/orhepatocellular carcinoma (HCC) and it is estimated that up to 40% ofchronically infected patients will develop at least one of thesecomplications.

The primary determinant of whether hepatitis B virus is cleared orestablishes a chronic infection is the robustness of the immuneresponse, in particular the CD8⁺ T cell response. Data from both animalmodels and infected patients indicate that strong innate immuneresponses are crucial in controlling initial HBV replication and forsubsequently activating the adaptive T cell response (reviewed inRehermann and Nascimbeni; Bertoletti and Gehring). In patients thatresolve acute infections, there are greater numbers of IFN-γ secretingCD4⁺ and CD8⁺ T cells with a broader range of epitope recognition thanin chronically infected patients (as reviewed in Bertoletti and Gehring;Desmond et al.). Although individuals that initially fail to mountvigorous T cell responses develop chronic infection, data indicate thatvirus specific T cells are still capable of a broad, effective T cellresponse. Rehermann et al. demonstrated that a small number ofchronically infected individuals mount robust CTL responses against HBVeither spontaneously or in response to IFN-α treatment. These T cellsare directed against multiple proteins indicating that chronicallyinfected patients can also mount a broad response to viral antigens.These data suggest that therapeutic interventions designed to stimulaterobust and multi-epitope specific responses may be sufficient to resolvechronic HBV infections. Yet, despite an effective prophylactic vaccine,there are currently no therapies capable of eliminating HBV fromchronically infected individuals. A number of anti-HBV therapeuticvaccines have been tested including traditional prophylactic vaccines,antigen/antibody complexes, lipopeptide, DNA, and recombinant virusbased strategies with limited success. Thus, there is a critical needfor more targeted therapeutic vaccines capable of inducing robust,sustained T cell responses capable of permanent clearance of virus.

Therapeutic peptide based vaccines are an attractive method for inducingCD4⁺ and CD8⁺ T cell responses in chronically infected individuals.While other vaccine formulations (such as DNA and recombinant virusvaccines) induce T cell responses, the peptide epitopes generated aftervaccination may not accurately reflect those generated during chronicinfection and therefore may not induce the necessary polyclonal responseneeded to clear the virus. In contrast, formulating a vaccine withmultiple epitopes presented by the chronically infected cells that arecapable of activating T cells to generate a polyclonal response wouldbypass the need for translation and processing of the parent protein andallow for expansion of the appropriate T cell specificities.

Peptide antigens for these early stage clinical studies were identifiedby motif prediction algorithms and selected by screening CTLs from acuteand chronically HBV infected patients. However, the T cell epitopespresented by HBV infected cells have not been reported or used in aclinical study.

Here we took an immunoproteomic approach to identify MHC class Ipeptides presented by chronic HBV infected cells. This approach hasdistinct advantages over traditional vaccine design algorithms as itidentifies antigens naturally processed and presented by infected, butnot healthy, cells. The identification of peptides and proteins derivedfrom HBV infection that are effectively recognized by the cellular armof the immune response forms the basis for a new and effective vaccine.Such peptides are displayed on the surface of infected cells inassociation with MHC class I and class II molecules and serve asrecognition targets for cytolytic and helper T cells of the immunesystem.

The present disclosure involves peptides that are associated with theHLA-A2, and HLA-A24 molecules, HLA-A2 supertypes, and HLA-A24supertypes. A supertype is a group of HLA molecules that present atleast one shared epitope. The present disclosure involves peptides thatare associated with HLA molecules, and with the genes and proteins fromwhich these peptides are derived.

Several methods have been developed to identify the peptides recognizedby CTL, each method relying on the ability of a CTL to recognize andkill only those cells expressing the appropriate class I MHC moleculewith the peptide bound to it. Such peptides can be derived from anon-self source, such as a pathogen (for example, following theinfection of a cell by a virus, such as HBV virus infection) or from aself-derived protein within a cell, such as a cancerous cell.

Three different methodologies have typically been used for identifyingthe peptides that are recognized by CTLs in infectious disease field.These are: (1) the genetic method; (2) motif analysis; (3) theimmunological and analytical chemistry methods or the Immunoproteomicsmethod. The genetic and motif prediction methodologies have typicallybeen used for identifying the peptides that are recognized by CTLs,which suffer from various drawbacks. A useful technique has been theimmunoproteomics method involving a combination of cellular immunologyand mass spectrometry. This approach involves the actual identificationof endogenous CTL epitopes present on the cell surface by sequencing thenaturally occurring peptides associated with class I MHC molecules. Inthis approach, cells are first lysed in a detergent solution, thepeptides associated with the class I MHC molecules are purified, and thepeptides are fractionated by high performance liquid chromatography(HPLC). Peptide sequencing is readily performed by tandem massspectrometry. The sequence can be confirmed by direct synthesis thereof(See Examples 4 and 5, below). Once confirmed such synthetic peptidescan be used to test their ability to activate CTLs against cellsinfected with the HBV virus.

A number of recent reports for different types of virus infectionsprovide evidence that CTL specific for epitopes that are naturallyprocessed and presented by infected cells have markedly greater impacton the control of virus replication. Undoubtedly, CTLs have been shownto play an important role in the elimination of HBV-infected cells.Thus, identification of antigenic peptides that are presented byinfected cells and recognized by epitope-specific CTLs may suggest newways to suppress viral replication and prevent persistent infection.Multiple peptides from conserved regions of HBV may prove essential inthe development of a universally immunogenic vaccine.

Little is known about cross genotypes conserved T cell epitopes that areimmunologically relevant in eliciting an effective T cell response tothe various HBV genotypes. Several groups have attempted to identify Tcell epitopes by either motif prediction of MHC binding peptides fromHBV proteins, or by screening overlapping peptides from structural andnonstructural viral proteins. Screening PBMCs from infected individualsusing a panel of algorithm-derived peptide sequences identified a fewcross genotype specific T cell epitopes. However, a comprehensiveanalysis of naturally presented epitopes on infected cells has neverbeen undertaken or reported.

Immunization with virus-derived, class I MHC-encoded molecule-associatedpeptides, or with a precursor polypeptide or protein that contains thepeptide, or with a gene that encodes a polypeptide or protein containingthe peptide, are forms of immunotherapy that can be employed in thetreatment of infections. These forms of immunotherapy require thatimmunogens be identified so that they can be formulated into anappropriate vaccine.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to Immunogens, such as polypeptides andfunctionally similar structures, comprising a novel epitopic peptidesequence of between 8 and 14, amino acids in length, most especially thesequence of SEQ ID NO: 1-17 and which immunogens facilitate a cytotoxicT lymphocyte (CTL)-mediated immune response against various strains ofHBV infected cells.

The present invention also relates to nucleic acid molecules that encodepolypeptides comprising said epitopic peptide, and which can also beused to facilitate an immune response against HBV infected cells.

The present invention provides compositions comprising the polypeptidesand immunogens described herein whereby the oligopeptides andpolypeptides of such immunogens are capable of inducing a CTL responseagainst cells expressing a protein comprising an epitopic sequence ofSEQ ID NO: 1-17 presented in association with Class I MHC protein, whichcells are infected with various strains of HBV.

In specific embodiments, the oligopeptides of the invention have asequence that comprises SEQ ID NO: 1-17 and are used as part of a largerstructure, most advantageously a polypeptide, including both naturallyoccurring polypeptides and synthetic polypeptides. The immunogens of theinvention incorporate such epitopic peptide sequences, either with suchsequences attached to form a larger antigenic structure or just as partof a polypeptide sequence incorporating such peptides as part of theamino acid sequence thereof in a pharmaceutical composition containingat least one immunogen and a pharmaceutically acceptable carrier.

Where the immunogens of the invention are polypeptides, or mixtures ofpolypeptides in the form of a pharmaceutically acceptable salt, suchpolypeptides can be of any length as long as part of their sequencecomprises at least one peptide of SEQ ID NO: 1-17, or sequence highlyhomologous thereto, ordinarily differing by no more than one amino acidresidue, including multiple copies of said sequence, when it is desiredto induce a CTL response against such peptide and thereby against HBVinfected cells.

The present invention further relates to polynucleotides comprising thegene coding for a polypeptide of the immunogens disclosed herein. Thepresent invention also provides methods that comprise contacting alymphocyte, especially a CTL, with an immunogen or its isoforms orsplice variants of the invention under conditions that induce a CTLresponse against various strains of HBV infected cell, and morespecifically HBV A infected cell. The methods may involve contacting theCTL with the immunogenic peptide in vivo, in which case the peptides,polypeptides, and polynucleotides of the invention are used as vaccines,and will be delivered as a pharmaceutical composition comprising apharmaceutically acceptable carrier or delivery system and theimmunogen, typically along with an adjuvant or one or more cytokines.

Alternatively, the immunogens of the present invention can be used toinduce a CTL response in vitro. The generated CTL can then be introducedinto a patient with HBV infection. Alternatively, the ability togenerate CTLs in vitro can serve as a diagnostic for HBV infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: HBV specific peptides stimulated CD8⁺ T cell activation invitro. (A.) HLA-A2 restricted CTLs directed against the identifiedpeptides (peptides are represented as first 3 residues of the sequence)were generated as previously described. PBMCs containing the epitopespecific CTLs were harvested, washed, and cultured with the peptidepulsed (A) or HBV expressing cells (B) overnight in an IFN-gamma ELISpotassay. Data is represented as % increase over background. (B.) PBMCscontaining epitope specific CTLs were harvested, washed, and culturedwith uninfected or HBV expressing cells overnight in an IFN-gammaELISpot assay. Normal liver cells served as a negative, non-specificcontrol.

FIG. 2: HBV identified peptides are able to activate CD8⁺ T cells invivo in both an HLA-A2 and HLA-A24 restricted fashion. HLA-A2 (A) orHLA-A24 (B) transgenic mice were primed and boosted with peptides aspreviously described. Spleens were harvested, homogenized into singlecell suspensions, and cultured with peptide pulsed (peptides arerepresented as first 3 residues of the sequence) HepG2 cells or HBVexpressing cells (DE19) overnight in an IFN-gamma ELISpot assay. T cellactivation was also measured by examining CD107a upregulation on HLA-A2(C) or HLA-A24 (D) CD8⁺ T cells. Splenocytes were cultured for 6 hourswith peptide pulsed or HBV expressing cells in the presence ofanti-CD107a and subsequently stained for CD8⁺ expression. Data ispresented as the percent of cells in culture that are CD8⁺ CD107a+.

FIG. 3: CD8⁺ T cells activated in vivo secrete cytotoxic effectormolecules. In an assay that mirrored the setup described in FIG. 2,splenocytes were cultured with peptide pulsed (peptides are representedas first 3 residues of the sequence) or HBV expressing cells (DE19 orHepG2 2.215) overnight. Supernatant was harvested and used in theMilliplex magnetic bead assay to detected granzyme B secretion inresponse to specific stimulation. Splenocytes from PBS primed, naïvemice were used as a negative control.

DETAILED SUMMARY OF THE INVENTION

As used herein and except as noted otherwise, all terms are defined asgiven below. The term “peptide” is used herein to designate a series ofamino acid residues, connected one to the other typically by peptidebonds between the alpha-amino and carbonyl groups of the adjacent aminoacids. The peptides are typically 9 amino acids in length, but can be asshort as 8 amino acids in length, and as long as 14 amino acids inlength. The series of amino acids are consider an “oligopeptide” whenthe amino acid length is greater than about 14 amino acids in length,typically up to about 30 to 40 residues in length. When the amino acidresidue length exceeds 40 amino acid residues, the series of amino acidresidues is termed “polypeptide”.

A peptide, oligopeptide, polypeptide, protein, or polynucleotide codingfor such a molecule is “immunogenic” and thus an immunogen within thepresent invention if it is capable of inducing an immune response. Inthe present invention, immunogenicity is more specifically defined asthe ability to induce a CTL-mediated response. Thus, an immunogen wouldbe a molecule that is capable of inducing an immune response, and in thepresent invention, a molecule capable of inducing a CTL response. Animmunogen may have one or more isoforms or splice variants that haveequivalent biological and immunological activity, and are thus alsoconsidered for the purposes of this invention to be immunogenicequivalents of the original, natural polypeptide.

A T cell “epitope” is a short peptide molecule that binds to a class Ior II MHC molecule and that is subsequently recognized by a T cell. Tcell epitopes that bind to class I MHC molecules are typically 8-14amino acids in length, and most typically 9 amino acids in length.

Three different genetic loci encode for class I MHC molecules: HLA-A,HLA-B, and HLA-C. The present invention involves peptides that areassociated with HLA-A2 supertypes. A supertype is a group of HLAmolecules that present at least one shared epitope. MHC moleculepeptides that have been found to bind to one member of the MHC allelesupertype family (A2 for example) are thought to be likely to bind toother members of the same supertype family (A68 for example).

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence.

The term “nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides. The nucleotide sequence encoding for a particularpeptide, oligopeptide, or polypeptide naturally occurring orsynthetically constructed.

The term “fragment,” when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region whoseexpression product retains essentially the same biological orimmunological function or activity as the expression product of thecomplete coding region.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring).

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form.

The term “active fragment” means a fragment that generates an immuneresponse (i.e., has immunogenic activity) when administered, alone oroptionally with a suitable adjuvant, to an animal, such as a mammal, forexample, a human, such immune response taking the form of stimulating aCTL response within the recipient. Alternatively, the “active fragment”may also be used to induce a CTL response in vitro.

As used herein, the terms “portion,” “segment,” and “fragment,” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, the oligopeptidesresulting from such treatment would represent portions, segments orfragments of the starting polypeptide.

The term “percent identity” when referring to a sequence, means that asequence is compared to a described sequence after alignment of thesequence to be compared with the described sequence. The PercentIdentity is determined according to the following formula:

Percent Identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequence(“R”) and the Compared Sequence (“C”) over the length of alignmentbetween R and C wherein (i) each base or amino acid in R that does nothave a corresponding aligned base or amino acid in the C and (ii) eachgap in R and (iii) each aligned base or amino acid in R that isdifferent from an aligned base or amino acid in C, constitutes adifference; and R is the number of bases or amino acids over the lengthof the alignment with C with any gap created in R also being counted asa base or amino acid.

The present invention relates generally to immunogens and immunogeniccompositions, and methods of use thereof, for the prevention, treatment,and diagnosis of HBV viral infections, especially HBV virus infection.Disclosed according to the invention are immunogens comprising proteinsor polypeptides whose amino acid sequences comprise one or more epitopicpeptides with sequences homologous to, preferably identical to, thesequence of SEQ ID NO: 1-17. In addition, the invention further relatesto polynucleotides that can be used to stimulate a CTL response againstHBV-infected cells, especially cells infected with the causativeorganism of HBV virus.

In accordance with the present invention there are disclosed specificamino acid sequences (SEQ ID NO: 1-17) which represent epitopic peptides(i.e. immunogenic peptide sequences) of at least about 8 amino acids inlength and no longer than about 14 amino acids in length and which arepresent as part of a larger structure, such as a polypeptide or fulllength protein, to form an immunogen of the invention. Proteins presentin the cells of HBV infection show these sequences. In addition,synthetic oligopeptides and polypeptides according to the invention alsocontain this sequence in one or more copies.

When the immunogens of the present invention comprise, or are formed of,polypeptides, these have amino acid sequences that comprise at least onestretch, possibly two, three, four, or more stretches of about 8 to 14residues in length and wherein any such segment within such sequencediffers in amino acid sequence from the sequence of SEQ ID NO: 1-17 byno more than about 1 amino acid residue, giving an overall sequenceidentity or homology of at least about 88%, preferably a conservativeamino acid residue, especially amino acids of the same general chemicalcharacter, such as where they are hydrophobic amino acids, or polaramino acids, or acidic amino acids or basic (alkaline) amino acids.

The present invention is also directed to an isolated polypeptide,especially one having immunogenic activity, the sequence of whichcomprises within it one or more stretches comprising any 2 or more ofthe sequences of SEQ ID NO: 1-17 and in any relative quantities andwherein said sequences may differ by one amino acid residues from thesequences of SEQ ID NO: 1-17 in any given stretch of 8 to 10, or up to14 amino acid residues. Thus, within the present invention, by way of anon-limiting example only, such polypeptide may contain as part of itsamino acid sequence, nanopeptide fragments having up to 8 amino acidsidentical to a sequence of SEQ ID NO: 1, 2, 7, 8 such that thepolypeptide comprises, in a specific embodiment, 2 segments with atleast 8 residues identical to SEQ ID NO: 1 and SEQ ID NO: 2 and onesegment with at least 8 residues identical to SEQ ID NO: 7. In otherembodiments, other combinations and permutations of the epitopicsequences disclosed herein may be part of an immunogen of the presentinvention or of such a polypeptide so long as any such polypeptidecomprises at least 2 such epitopes, whether such epitopes are differentor the same. Thus, in a specific embodiment, a polypeptide of thepresent invention may comprise 2 copies of the sequence of SEQ ID NO: 2at some point or points within its length. Of course, any combinationsand permutations of the epitopes disclosed herein, as long as they arepresent at least two in number in such polypeptides, are expresslycontemplated.

Peptides of the invention are commonly immunogens, or at least can haveimmunogenic activity, possibly requiring a larger carrier molecule tofacilitate such activity, or said peptides may have immunogenic activitywhen part of a larger structure, such as a polypeptide, other than theprotein found in the HBV organism itself. Such peptides may also haveimmunogenic activity when part of a composition containing one or moreof said epitopic peptides, which may be present in any combination andwith each such peptide being present in one or more copies.

Said polypeptides can be of any desired length so long as they haveimmunogenic activity in that they are able, under a given set ofdesirable conditions, to elicit in vitro or in vivo the activation ofcytotoxic T lymphocytes (CTLs) (i.e., a CTL response) against apresentation of a HBV-infected cell, and when such proteins arepresented along with MHC-1 proteins, such as where said proteins arepresented in vitro or in vivo by an antigen presenting cell (APC). Theproteins and polypeptides forming the immunogens of the presentinvention can be naturally occurring or may be synthesized chemically.

The epitopic sequence (SEQ ID NO: 1-17) present within polypeptides andproteins forming the immunogens of the present invention includesequences as short as 7, preferably 8, amino acid residues and as longas 15, preferably 14, amino acids in length. The present invention alsoencompasses peptides at least about 88% identical to the peptides orsequences of SEQ ID NO: 1-17 disclosed herein and to sequences differingfrom these sequences by no more than one amino acid, including fragmentscontaining sequences having at least 8 residues in common with thesequences of SEQ ID NO: 1-17 over any nine residue length and whereinsaid homologous sequence of residues need not be continuous so that saidlength may contain up to one amino acid not in common with the sequenceof SEQ ID NO: 1-17 or be identical to said sequence but include oneadditional residue or have one less residue relative to said sequenceand whereby such different amino acid unit or residue may occur anywherewithin the corresponding stretch within said immunogen or polypeptide.

The present invention is also directed to an isolated polypeptide,including a purified polypeptide, especially one having immunogenicactivity, the sequence of which comprises within it one or more copiesof epitopic peptide sequences homologous, if not identical, to thesequence of SEQ ID NO: 1-17 and wherein said sequences may differ by oneamino acid residues from the sequence of SEQ ID NO: 1-17. Thus, withinthe present invention, such polypeptide may contain as part of its aminoacid sequence, oligopeptides having up to 8 amino acids in length anddiffering by no more than one amino acid residue as compared to thesequence of SEQ ID NO: 1-17 such that the polypeptide comprises, in onespecific embodiment, 2 segments each with a sequence differing by nomore than one amino acid residue from SEQ ID NO: 1-17 and 1 segmentidentical to SEQ ID NO: 1-17. In other embodiments, other combinationsand permutations of the epitopic sequence disclosed herein may be partof an immunogen of the present invention or of such a polypeptide solong as any such polypeptide comprises at least 2 such epitopes, whethersuch epitopes are identical or differ by a residue. In other preferredembodiments, such immunogen, especially where a polypeptide, maycomprise as part of its amino acid sequence, a number of oligopeptidesegments as disclosed herein such that there are 2, 3, 4, 5, or moresuch segments and wherein such segments are contiguous or are notcontiguous or where some are contiguous and some are not contiguous.

The present invention further relates to isolated oligopeptides of atleast 8 but not more than 14 amino acid units in length and having asequence differing at most by no more than one amino acid residue from asequence selected from the group consisting of the sequence of SEQ IDNO: 1-17. Thus, the present invention relates to a immunogen comprisinga peptide segment of at least 8 but not more than 14 amino acid units inlength which segment comprises a sequence selected from the groupconsisting of the sequence of SEQ ID NO: 1-17 or a sequence differingfrom said sequence by not more than 1 amino acid.

In preferred embodiments, where the isolated oligopeptides of theinvention are homologous to the sequences of SEQ ID NO: 1-17, saiddifference of one amino acid residue is the result of a substitution ofone hydrophobic amino acid unit by another hydrophobic amino acid, or isthe result of a substitution of one polar amino acid unit by anotherpolar amino acid, or is a substitution of one acidic amino acid unit byanother acidic amino acid, or is the result of a substitution of onebasic amino acid unit by another basic amino acid.

The present invention further relates to a composition comprising one ormore of the isolated oligopeptides of the invention suspended in apharmacologically acceptable carrier or vaccine delivery vehicle.

Oligopeptides as disclosed herein may themselves be prepared by methodswell known to those skilled in the art. (Grant, G. A., SyntheticPeptides: A User's Guide, 1792, W. H. Freeman and Company, New York;Coligan, J. E. et al, Current Protocols in Protein Science, 1799, JohnWiley & Sons, Inc., New York).

Besides the sequences of SEQ ID NO: 1-17, the proteins and polypeptidesforming the immunogens of the present invention may also comprise one ormore other immunogenic amino acid stretches known to be associated withvarious strains of HBV infection, and which may stimulate or enhance aCTL response whereby the immunogenic peptides associate with HLA-A2 orA24 supertypes or another class I MHC (i.e., MHC-1) molecule.

The immunogens of the present invention can be in the form of acomposition of one or more of the different immunogens and wherein eachimmunogen is present in any desired relative abundance. Suchcompositions can be homogeneous or heterogeneous with respect to theindividual immunogens or polypeptides of the invention, or theimmunogenic peptide components present in such polypeptides or proteinsor immunogens, having only one or more than one of such peptides. Forexample, an isolated peptide of the present invention can have thesequence of SEQ ID NO: 1-17 or differ there from by 1 amino acid andsuch peptides can be used to form an immunogenic composition of saidpeptides as already disclosed herein.

The oligopeptides and polypeptides useful in practicing the presentinvention may be derived by fractionation of naturally occurringproteins by methods such as protease treatment, or they may be producedby recombinant or synthetic methodologies that are well known and clearto the skilled artisan (Ausubel, F. M. et al, Current Protocols inMolecular Biology, 1799, John Wiley & Sons, Inc., New York; Coligan, J.E. et al, Current Protocols in Protein Science, 1799, John Wiley & Sons,Inc., New York; Molecular Cloning: A Laboratory Manual, 1789, ColdSpring Harbor Laboratory Press, Cold Spring Harbor). The polypeptide maycomprise a recombinant or synthetic polypeptide that comprises at leastone of SEQ ID NO: 1-17 which sequences may also be present in multiplecopies. Thus, oligopeptides and polypeptides of the present inventionmay have one, two, three, or more such immunogenic peptides within theamino acid sequence of said oligopeptides and polypeptides, and saidimmunogenic peptides, or epitopes, may be the same or may be different,or may have any number of such sequences wherein some of them areidentical to each other in amino acid sequence while others within thesame polypeptide sequence are different from each other and saidepitopic sequences may occur in any order within said immunogenicpolypeptide sequence. The location of such sequences within the sequenceof a polypeptide forming an immunogen of the invention may affectrelative immunogenic activity. In addition, immunogens of the presentinvention may comprise more than one protein comprising the amino acidsequences disclosed herein. Such polypeptides may be part of a singlecomposition or may themselves be covalently or non-covalently linked toeach other.

Where the immunogen comprises two or more immunogenic epitopes, orepitopic peptides, they may be linked directly together, or through aspacer or linker, to form a larger structure, such as an oligopeptide,or polypeptide, or some other polymeric structure. The epitopic peptidesmay therefore be linked by any and all means that can be devised by thechemist so long as the immunogenic activity of the overall structure orcomplex is maintained or, at least, not reduced below a level useful forthe methods of the invention (i.e., especially where said immunogenicactivity comprises being capable of eliciting a CTL response).

Likewise, the immunogenic peptides disclosed herein may also be linkeddirectly to, or through, a spacer or linker to: an immunogenic carriersuch as serum albumin, tetanus toxoid, keyhole limpet hemocyanin,dextran, or a recombinant virus particle or any synthetic nanoparticlessuch as liposimes, polymers and metal such as gold nanoparticles; animmunogenic peptide known to stimulate a T helper cell type immuneresponse; a cytokine such as interferon gamma or GMCSF(Granulocyte-Monocyte Colony Stimulating Factor); a targeting agent suchas an antibody or receptor ligand; a stabilizing agent such as a lipid;or a conjugate of a plurality of epitopes to a branched lysine corestructure, such as the so-called “multiple antigenic peptide” describedin (Posnett, D. N. et al., J. Biol. Chem., 263:1717-1725, (1788)); acompound such as polyethylene glycol to increase the half life of thepeptide; or additional amino acids such as a leader or secretorysequence, or a sequence employed for the purification of the maturesequence.

Useful spacers and linkers are typically comprised of relatively small,neutral molecules, such as amino acids and which are substantiallyuncharged under physiological conditions. Such spacers are typicallyselected from the group of nonpolar or neutral polar amino acids, suchas glycine, alanine, serine and other similar amino acids. Such optionalspacers or linkers need not be comprised of the same residues and thusmay be either homo- or hetero-oligomers. When present, such linkers willcommonly be of length at least one or two, commonly 3, 4, 5, 6, andpossibly as much as 10 or even up to 20 residues (in the case of aminoacids). In addition, such linkers need not be composed of amino acidsbut any oligomeric structures will do as well so long as they providethe correct spacing so as to optimize the desired level of immunogenicactivity of the immunogens of the present invention. The immunogen maytherefore take any form that is capable of eliciting a CTL response.

In addition, the immunogenic peptides of the present invention may bepart of an immunogenic structure via attachments other than conventionalpeptide bonds. Thus, any manner of attaching the peptides of theinvention to an immunogen of the invention, such as an immunogenicpolypeptide as disclosed herein, could provide an immunogenic structureas claimed herein. Thus, immunogens, such as proteins of the invention,are structures that contain the peptides disclosed according to thepresent invention but such immunogenic peptides may not necessarily beattached thereto by the conventional means of using ordinary peptidebounds. The immunogens of the present invention simply contain suchpeptides as part of their makeup, but how such peptides are to becombined to form the final immunogen is left to the talent andimagination of the user and is in no way restricted or limited by thedisclosure contained herein.

The peptides that are naturally processed and bound to a class I MHCmolecule in accordance with the invention need not be the optimalpeptides for stimulating a CTL response. See, for example, (Parkhurst,M. R. et al., J. Immunol., 157:2539-2548, (1796); Rosenberg, S. A. etal., Nat. Med., 4:321-327, (1798)). Thus, there can be utility inmodifying a peptide, such that it more readily induces a CTL response.Generally, peptides may be modified at two types of positions. Thepeptides may be modified at amino acid residues that are predicted tointeract with the class I MHC molecule, in which case the goal is tocreate a peptide that has a higher affinity for the class I MHC moleculethan does the parent peptide. The peptides can also be modified at aminoacid residues that are predicted to interact with the T cell receptor onthe CTL, in which case the goal is to create a peptide that has a higheraffinity for the T cell receptor than does the parent peptide. Both ofthese types of modifications can result in a variant peptide that isrelated to a parent peptide, but which is better able to induce a CTLresponse than is the parent peptide. As used herein, the term “parentpeptide” means an oligopeptide having the sequence of SEQ ID NO: 1-17.

The parent peptides disclosed herein can be modified by the substitutionof one or more residues at different, possibly selective, sites withinthe peptide chain. Such substitutions may be of a conservative nature,for example, where one amino acid is replaced by an amino acid ofsimilar structure and characteristics, such as where a hydrophobic aminoacid is replaced by another hydrophobic amino acid. Even moreconservative would be replacement of amino acids of the same or similarsize and chemical nature, such as where leucine is replaced byisoleucine. In studies of sequence variations in families of naturallyoccurring homologous proteins, certain amino acid substitutions are moreoften tolerated than others, and these are often show correlation withsimilarities in size, charge, polarity, and hydrophobicity between theoriginal amino acid and its replacement, and such is the basis fordefining “conservative substitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1—small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2—polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3—polar,positively charged residues (His, Arg, Lys); Group 4—large, aliphatic,nonpolar residues (Met, Leu, lie, Val, Cys); and Group 4—large, aromaticresidues (Phe, Tyr, Trp). An acidic amino acid might also be substitutedby a different acidic amino acid or a basic (i.e., alkaline) amino acidby a different basic amino acid.

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such radical substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,amino acids possessing non-standard R groups (i.e., R groups other thanthose found in the common 20 amino acids of natural proteins) may alsobe used for substitution purposes to produce immunogens and immunogenicpolypeptides according to the present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsyngeneic effects on the antigenicity of the peptide. At most, no morethan 1 position (possibly 2 positions) within the peptide wouldsimultaneously be substituted.

Based on cytotoxicity assays, an epitope is considered substantiallyidentical to the reference peptide if it has at least 10% of theantigenic activity of the reference peptide as defined by the ability ofthe substituted peptide to reconstitute the epitope recognized by a CTLin comparison to the reference peptide. Thus, when comparing the lyticactivity in the linear portion of the effector:target curves withequimolar concentrations of the reference and substituted peptides, theobserved percent specific killing of the target cells incubated with thesubstituted peptide should be equal to that of the reference peptide atan effector:target ratio that is no greater than 10-fold above thereference peptide effector:target ratio at which the comparison is beingmade.

Preferably, when the CTLs specific for an oligopeptide of the inventionis tested against the substituted peptides, the peptide concentration atwhich the substituted peptides achieve half the maximal increase inlysis relative to background is no more than about 1 mM, preferably nomore than about 1 μM, more preferably no more than about 1 nM, and stillmore preferably no more than about 100 pM, and most preferably no morethan about 10 pM. It is also preferred that the substituted peptide berecognized by CTLs from more than one individual, at least two, and morepreferably three individuals.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than 4 residues from thereference peptide, as long as they have substantially identicalantigenic activity.

It should be appreciated that an immunogen may consist only of a peptideof SEQ ID NO: 1-17, or comprise a peptide of SEQ ID NO: 1-17, orcomprise a plurality of peptides selected from SEQ ID NO: 1-17, orcomprise a polypeptide that itself comprises one or more of the epitopicpeptides of SEQ ID NO: 1-17.

The immunogenic peptides and polypeptides of the invention can beprepared synthetically, by recombinant DNA technology, or they can beisolated from natural sources such as tumor cells expressing theoriginal protein product.

The polypeptides and oligopeptides disclosed herein can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automated peptide synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, (Grant, G. A., Synthetic Peptides: A User's Guide, 1792, W. H.Freeman and Company, New York; Coligan, J. E. et al, Current Protocolsin Protein Science, 1799, John Wiley & Sons, Inc., New York). Fragmentsof polypeptides of the invention can also be synthesized asintermediates in the synthesis of a larger polypeptide.

Recombinant DNA technology may be employed wherein a nucleotide sequencewhich encodes an immunogenic peptide or polypeptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell, and cultivated under conditions suitable forexpression. These procedures are well known in the art to the skilledartisan, as described in (Coligan, J. E. et al, Current Protocols inImmunology, 1799, John Wiley & Sons, Inc., New York; Ausubel, F. M. etal, Current Protocols in Molecular Biology, 1799, John Wiley & Sons,Inc., New York; Molecular Cloning: A Laboratory Manual, 1789, ColdSpring Harbor Laboratory Press, Cold Spring Harbor). Thus, recombinantlyproduced peptides or polypeptides can be used as the immunogens of theinvention.

The coding sequences for peptides of the length contemplated herein canbe synthesized on commercially available automated DNA synthesizersusing protocols that are well known in the art. See for example, (Grant,G. A., Synthetic Peptides: A User's Guide, 1792, W. H. Freeman andCompany, New York; Coligan, J. E. et al, Current Protocols in ProteinScience, 1799, John Wiley & Sons, Inc., New York). The coding sequencescan also be modified such that a peptide or polypeptide will be producedthat incorporates a desired amino acid substitution. The coding sequencecan be provided with appropriate linkers, be ligated into suitableexpression vectors that are commonly available in the art, and theresulting DNA or RNA molecule can be transformed or transfected intosuitable hosts to produce the desired fusion protein. A number of suchvectors and suitable host systems are available, and their selection isleft to the skilled artisan. For expression of the fusion proteins, thecoding sequence will be provided with operably linked start and stopcodons, promoter and terminator regions, and a replication system toprovide an expression vector for expression in the desired host cell.For example, promoter sequences compatible with bacterial hosts areprovided in plasmids containing convenient restriction sites forinsertion of the desired coding sequence. The resulting expressionvectors are transformed into suitable bacterial hosts. Of course, yeast,insect, and mammalian host cells may also be used, employing suitablevectors and control sequences.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Ausubel, F. M. et al, CurrentProtocols in Molecular Biology, 1799, John Wiley & Sons, Inc., New York;Molecular Cloning: A Laboratory Manual, 1789, Cold Spring HarborLaboratory Press, Cold Spring Harbor). Such cells can routinely beutilized for assaying CTL activity by having said geneticallyengineered, or recombinant, host cells express the immunogenic peptidesof the present invention.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1781), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The immunogenic peptides of the present invention may be used to elicitCTLs ex vivo from either healthy individuals or from patients with HBVinfection (or at risk thereof). Such responses are induced by incubatingin tissue culture the individual's CTL precursor lymphocytes togetherwith a source of antigen presenting cells and the appropriateimmunogenic peptide. Examples of suitable antigen presenting cellsinclude dendritic cells, macrophages, and activated B cells. Typically,the peptide at concentrations between 10 and 40 .mu.g/ml, would bepre-incubated with the antigen presenting cells for periods ranging from1 to 18 hrs. .beta..sub.2-microglobulin (4 .mu.g/ml) can be added duringthis time period to enhance binding. The antigen presenting cells mayalso be held at room temperature during the incubation period(Ljunggren, H.-G. et al., Nature, 346:476-480, (1790)) or pretreatedwith acid (Zeh, H. J., III et al., Hum. Immunol., 39:79-86, (1794)) topromote the generation of denatured class I MHC molecules which can thenbind the peptide. The precursor CTLs (responders) are then added to theantigen presenting cells to which the immunogenic peptide has bound(stimulators) at responder to stimulator ratios of between 5:1 and 50:1,and most typically between 10:1 and 20:1. The co-cultivation of thecells is carried out at 37.degree. C. in RPMI 1640, 10% fetal bovineserum, 2 mM L-glutamine, and IL-2 (5-20 Units/ml). Other cytokines, suchas IL-1, IL-7, and IL-12 may also be added to the culture. FreshIL-2-containing media is added to the cultures every 2-4 days, typicallyby removing one-half the old media and replenishing it with an equalvolume of fresh media. After 7-10 days, and every 7-10 days thereafter,the CTL are restimulated with antigen presenting cells to whichimmunogenic peptide has been bound as described above. FreshIL-2-containing media is added to the cells throughout their culture asdescribed above. Three to four rounds of stimulation, and sometimes asmany five to eight rounds of stimulation, are required to generate a CTLresponse that can then be measured in vitro. The above describedprotocol is illustrative only and should not be considered limiting.Many in vitro CTL stimulation protocols have been described and thechoice of which one to use is well within the knowledge of the skilledartisan. The peptide-specific CTL can be further expanded to largenumbers by treatment with anti-CD3 antibody. For example, see (Riddell,S. R. and Greenberg, P. D., J. Immunol. Methods, 128:189-201, (1790);Walter, E. A. et al., N. Engl. J. Med., 333:1038-1044, (1795)).

Antigen presenting cells that are to be used to stimulate a CTL responseare typically incubated with peptide of an optimal length as disclosedherein that allows for direct binding of the peptide to the class I MHCmolecule without additional processing. Larger oligopeptides andpolypeptides are generally ineffective in binding to class I MHCmolecules as they are not efficiently processed into an appropriatelysized peptide in the extracellular milieu. There are a variety ofapproaches that are known in the art, however, that allow oligopeptidesand polypeptides to be exogenously acquired by a cell, which then allowsfor their subsequent processing and presentation by a class I MHCmolecule. Representative, but non-limiting examples of such approachesinclude electroporation of the molecules into the cell (Harding, C. H.III, Eur. J. Immunol., 22:1865-1869, (1792)), encapsulation of themolecules in liposomes which are fused to the cells of interest (Reddy,R. et al., J. Immunol. Methods, 141:157-163, (1791)), or osmotic shockin which the molecules are taken up via pinocytosis (Moore, M. W. etal., Cell, 54:777-717, (1788)). Thus, oligopeptides and polypeptidesthat comprise one or more of the peptides of the invention can beprovided to antigen presenting cells in such a fashion that they aredelivered to the cytoplasm of the cell, and are subsequently processedto allow presentation of the peptides.

Antigen presenting cells suitable for stimulating an in vitro CTLresponse that is specific for one or more of the peptides of theinvention can also be prepared by introducing polynucleotide vectorsencoding the sequences into the cells. These polynucleotides can bedesigned such that they express only a single peptide of the invention,multiple peptides of the invention, or even a plurality of peptides ofthe invention. There are a variety of approaches that are known in theart, that allow polynucleotides to be introduced and expressed in acell, thus providing one or more peptides of the invention to the classI MHC molecule binding pathway. Representative, but non-limitingexamples of such approaches include the introduction of plasmid DNAthrough particle-mediated gene transfer or electroporation (Tuting, T.et al., J. Immunol., 160:1139-1147, (1798)), or the transduction ofcells with an adenovirus expressing the polynucleotide of interest(Perez-Diez, A. et al., Cancer Res., 58:5305-5309, (1798)). Thus,oligonucleotides that code for one or more of the peptides of theinvention can be provided to antigen presenting cells in such a fashionthat the peptides associate with class I MHC molecules and are presentedon the surface of the antigen presenting cell, and consequently areavailable to stimulate a CTL response.

By preparing the stimulator cells used to generate an in vitro CTLresponse in different ways, it is possible to control the peptidespecificity of CTL response. For example, the CTLs generated with aparticular peptide will necessarily be specific for that peptide.Likewise, CTLs that are generated with a polypeptide or polynucleotideexpressing or coding for particular peptides will be limited tospecificities that recognize those peptides. More broadly, stimulatorcells, and more specifically dendritic cells, can be incubated in thepresence of the whole parent protein. As a further alternative,stimulator cells, and more specifically dendritic cells, can betransduced or transfected with RNA or DNA comprising the polynucleotidesequence encoding the protein. Under these alternative conditions,peptide epitopes that are naturally cleaved out of the protein, andwhich are generated in addition to peptide epitopes of SEQ ID NO: 1-17can associate with an appropriate class I MHC molecule, which may or maynot include HLA-A1, -A2, -A24. The selection of antigen presenting cellsand the type of antigen with which to stimulate the CTL, is left to theordinary skilled artisan.

In certain embodiments, the methods of the present invention include amethod for inducing a CTL response in vitro that is specific for a tumorcell expressing a molecule from A1, A2, or A24 supertypes, whereby themethod comprises contacting a CTL precursor lymphocyte with an antigenpresenting cell that has bound an immunogen comprising one or more ofthe peptides disclosed according to the invention.

In specific embodiments, the methods of the present invention include amethod for inducing a CTL response in vitro that is specific for a tumorcell expressing a molecule from A1, A2, or A24 supertypes, whereby themethod comprises contacting a CTL precursor lymphocyte with an antigenpresenting cell that has exogenously acquired an immunogenicoligopeptide or polypeptide that comprises one or more of the peptidesdisclosed according to the invention.

A yet additional embodiment of the present invention is directed to aprocess for inducing a CTL response in vitro that is specific for atumor cell expressing a molecule from A1, A2, or A24 supertypes,comprising contacting a CTL precursor lymphocyte with an antigenpresenting cell that is expressing a polynucleotide coding for apolypeptide of the invention and wherein said polynucleotide is operablylinked to a promoter.

A variety of techniques exist for assaying the activity of CTL. Thesetechniques include the labeling of target cells with radionuclides suchas Na.sub.2.sup.51CrO.sub.4 or .sup.3H-thymidine, and measuring therelease or retention of the radionuclides from the target cells as anindex of cell death. Such assays are well-known in the art and theirselection is left to the skilled artisan. Alternatively, CTLs are knownto release a variety of cytokines when they are stimulated by anappropriate target cell, such as a cell expressing the relevant class IMHC molecule and the corresponding peptide. Non-limiting examples ofsuch cytokines include IFN-.gamma., TNF.alpha., and GM-CSF. Assays forthese cytokines are well known in the art, and their selection is leftto the skilled artisan. Methodology for measuring both target cell deathand cytokine release as a measure of CTL reactivity are given in(Coligan, J. E. et al, Current Protocols in Immunology, 1799, John Wiley& Sons, Inc., New York).

After expansion of the antigen-specific CTLs, the latter are thenadoptively transferred back into the patient, where they will destroytheir specific target cell, especially macrophages infected with M. HBVinfection. The utility of such adoptive transfer is demonstrated in(North, R. J. et al., Infect. Immun., 67:2010-2012, (1799); Riddell, S.R. et al., Science, 257:238-241, (1792)). In determining the amount ofcells to re-infuse, the skilled physician will be guided by the totalnumber of cells available, the activity of the CTL as measured in vitro,and the condition of the patient. Preferably, however, about1.times.10.sup.6 to about 1.times.10.sup.12, more preferably about1.times.10.sup.8 to about 1.times.10.sup.11, and even more preferably,about 1.times.10.sup.9 to about 1.times.10.sup.10 peptide-specific CTLare infused. Methodology for re-infusing the T cells into a patient arewell known and exemplified in U.S. Pat. No. 4,844,893 to Honski, et al.,and U.S. Pat. No. 4,690,915 to Rosenberg.

The peptide-specific CTL can be purified from the stimulator cells priorto infusion into the patient. For example, monoclonal antibodiesdirected towards the cell surface protein CD8, present on CTL, can beused in conjunction with a variety of isolation techniques such asantibody panning, flow cytometric sorting, and magnetic bead separationto purify the peptide-specific CTL away from any remaining non-peptidespecific lymphocytes or from the stimulator cells. These methods arewell known in the art, and are their selection is left to the skilledartisan. It should be appreciated that generation of peptide-specificCTL in this manner, obviates the need for stimulating the CTL in thepresence of tubercle-infected cells. Thus, there is no chance ofinadvertently reintroducing infected cells into the patient.

Thus, one embodiment of the present invention relates to a process fortreating a subject with cancer characterized by tumor cells expressingcomplexes of a molecule from A1, A2, or A24 supertypes, for example,HLA-A1, HLA-A2, or HLA-A24, whereby CTLs produced in vitro according tothe present invention are administered in an amount sufficient todestroy the tumor cells through direct lysis or to effect thedestruction of the tumor cells indirectly through the elaboration ofcytokines.

Another embodiment of the present invention is directed to a process fortreating a subject with cancer characterized by tumor cells expressingany class I MHC molecule and an epitope of SEQ ID NO: 1-17, whereby theCTLs are produced in vitro and are specific for the epitope or originalprotein and are administered in an amount sufficient to destroy thetumor cells through direct lysis or to effect the destruction of thetumor cells indirectly through the elaboration of cytokines.

In additional embodiments, ex vivo generated CTLs can be used toidentify and isolate the T cell receptor molecules specific for thepeptide. The genes encoding the alpha and beta chains of the T cellreceptor can be cloned into an expression vector system and transferredand expressed in nave T cells from peripheral blood, T cells from lymphnodes, or T lymphocyte progenitor cells from bone marrow. These T cells,which would then be expressing a peptide-specific T cell receptor, wouldthen have specific cytotoxic reactivity and could be used in adoptivetherapy to destroy HBV infected cells.

In addition to their use for therapeutic or prophylactic purposes, theimmunogenic peptides of the present invention are useful as screeningand diagnostic agents. Thus, the immunogenic peptides of the presentinvention, together with modem techniques of gene screening, make itpossible to screen patients for the presence of genes encoding suchpeptides on cells obtained from patients suspected of infection andpossibly the results of such screening may help determine the efficacyof HBV vaccines for protection against various strains of HBV infection.Proceeding with the regimen of treatment disclosed herein using theimmunogens of the present invention.

Alternatively, the immunogenic peptides disclosed herein, as well asfunctionally similar homologs thereof, may be used to screen a samplefor the presence of CTLs that specifically recognize the correspondingepitopes. The lymphocytes to be screened in this assay will normally beobtained from the peripheral blood, but lymphocytes can be obtained fromother sources, including lymph nodes, spleen, and pleural fluid. Thepeptides of the present invention may then be used as a diagnostic toolto evaluate the efficacy of the immunotherapeutic treatments disclosedherein. Thus, the in vitro generation of CTLs as described above wouldbe used to determine if patients are likely to respond to the peptide invivo. Similarly, the in vitro generation of CTLs could be done withsamples of lymphocytes obtained from the patient before and aftertreatment with the peptides and other immunogens of the invention.Successful generation of CTLs in vivo should then be recognized by acorrespondingly easier ability to generate peptide-specific CTLs invitro from lymphocytes obtained following treatment in comparison tothose obtained before treatment.

The oligopeptides of the invention, such as SEQ ID NO: 1-17, can also beused to prepare class I MHC tetramers which can be used in conjunctionwith flow cytometry to quantitate the frequency of peptide-specific CTLthat are present in a sample of lymphocytes from an individual.Specifically, for example, class I MHC molecules comprising HLA-A2 andpeptides highly homologous, meaning differing by 1 amino acid residue,including where, for example, the peptide sequence has 8 or 10 residues,to SEQ ID NO:1 would be combined to form tetramers as exemplified inU.S. Pat. No. 5,635,363. Said tetramers would find use in monitoring thefrequency of CTLs in the peripheral blood, lymph nodes, or tumor mass ofan individual undergoing immunotherapy with the peptides, proteins, orpolynucleotides of the invention, and it would be expected thatsuccessful immunization would lead to an increase in the frequency ofthe peptide-specific CTL.

As stated above, a vaccine in accordance with the present invention mayinclude one or more of the hereinabove described polypeptides or activefragments thereof, or a composition, or pool, of immunogenic peptidesdisclosed herein. When employing more than one polypeptide or activefragment, such as two or more polypeptides and/or active fragments maybe used as a physical mixture or as a fusion of two or more polypeptidesor active fragments. The fusion fragment or fusion polypeptide may beproduced, for example, by recombinant techniques or by the use ofappropriate linkers for fusing previously prepared polypeptides oractive fragments.

The immunogenic molecules of the invention, including vaccinecompositions, may be utilized according to the present invention forpurposes of preventing, suppressing or treating diseases causing theexpression of the immunogenic peptides disclosed herein, such as wherethe antigen is being expressed by HBV infected cells. As used inaccordance with the present invention, the term “prevention” relates toa process of prophylaxis in which an animal, especially a mammal, andmost especially a human, is exposed to an immunogen of the presentinvention prior to the induction or onset of the disease process. Thus,the immunogen could be administered to the general population as isfrequently done for infectious diseases. Alternatively, the term“suppression” is often used to describe a condition wherein the diseaseprocess has already begun but obvious symptoms of said condition haveyet to be realized. Thus, the cells of an individual may have becomeinfected but no outside signs of the disease have yet been clinicallyrecognized. In either case, the term prophylaxis can be applied toencompass both prevention and suppression. Conversely, the term“treatment” is often utilized to mean the clinical application of agentsto combat an already existing condition whose clinical presentation hasalready been realized in a patient. This would occur where an individualhas already been diagnosed as having an HBV infection.

It is understood that the suitable dosage of an immunogen of the presentinvention will depend upon the age, sex, health, and weight of therecipient, the kind of concurrent treatment, if any, the frequency oftreatment, and the nature of the effect desired. However, the mostpreferred dosage can be tailored to the individual subject, asdetermined by the researcher or clinician. The total dose required forany given treatment will commonly be determined with respect to astandard reference dose as set by a manufacturer, such as is commonlydone with vaccines, such dose being administered either in a singletreatment or in a series of doses, the success of which will depend onthe production of a desired immunological result (i.e., successfulproduction of a CTL-mediated response to the antigen, which responsegives rise to the prevention and/or treatment desired). Thus, theoverall administration schedule must be considered in determining thesuccess of a course of treatment and not whether a single dose, given inisolation, would or would not produce the desired immunologicallytherapeutic result or effect.

The therapeutically effective amount of a composition containing one ormore of the immunogens of this invention is an amount sufficient toinduce an effective CTL response to the antigen and to cure or arrestdisease progression. Thus, this dose will depend, among other things, onthe identity of the immunogens used, the nature of the diseasecondition, the severity of the disease condition, the extent of any needto prevent such a condition where it has not already been detected, themanner of administration dictated by the situation requiring suchadministration, the weight and state of health of the individualreceiving such administration, and the sound judgment of the clinicianor researcher. Thus, for purposes of prophylactic or therapeuticadministration, effective amounts would generally lie within the rangeof from 1.0 μg to about 5,000 μg of peptide for a 70 kg patient,followed by boosting dosages of from about 1.0 μg to about 1,000 μg ofpeptide pursuant to a boosting regimen over days, weeks or even months,depending on the recipient's response and as necessitated by subsequentmonitoring of CTL-mediated activity within the bloodstream. Of course,such dosages are to be considered only a general guide and, in a givensituation, may greatly exceed such suggested dosage regimens where theclinician believes that the recipient's condition warrants moreaggressive administration schedule. Needless to say, the efficacy ofadministering additional doses, and of increasing or decreasing theinterval, may be re-evaluated on a continuing basis, in view of therecipient's immunocompetence.

For such purposes, the immunogenic compositions according to the presentinvention may be used against a disease condition such as HBV infectionby administration to an individual by a variety of routes. Thecomposition may be administered parenterally or orally, and, ifparenterally, either systemically or topically. Parenteral routesinclude subcutaneous, intravenous, intradermal, intramuscular,intraperitoneal, intranasal, transdermal, or buccal routes. One or moresuch routes may be employed. Parenteral administration can be, forexample, by bolus injection or by gradual perfusion over time.

Generally, vaccines are prepared as injectables, in the form of aqueoussolutions or suspensions. Vaccines in an oil base are also well knownsuch as for inhaling. Solid forms which are dissolved or suspended priorto use may also be formulated. Pharmaceutical carriers, diluents andexcipients are generally added that are compatible with the activeingredients and acceptable for pharmaceutical use. Examples of suchcarriers include, but are not limited to, water, saline solutions,dextrose, or glycerol. Combinations of carriers may also be used. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques including sterile filtration. The resulting solutions may bepackaged for use as is, or the aqueous solutions may be lyophilized, thelyophilized preparation being combined with sterile water beforeadministration. Vaccine compositions may further incorporate additionalsubstances to stabilize pH, or to function as adjuvants, wetting agents,or emulsifying agents, which can serve to improve the effectiveness ofthe vaccine.

The concentration of the CTL stimulatory peptides of the invention inpharmaceutical formulations are subject to wide variation, includinganywhere from less than 0.01% by weight to as much as 50% or more.Factors such as volume and viscosity of the resulting composition mustalso be considered. The solvents, or diluents, used for suchcompositions include water, possibly PBS (phosphate buffered saline), orsaline itself, or other possible carriers or excipients.

The immunogens of the present invention may also be contained inartificially created structures such as liposomes, ISCOMS,slow-releasing nanoparticles, and other vehicles which increase theimmunogenicity and/or half-life of the peptides or polypeptides inserum. Liposomes include emulsions, foams, micelles, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like. Liposomes for use in the invention are formed fromstandard vesicle-forming lipids which generally include neutral andnegatively charged phospholipids and a sterol, such as cholesterol. Theselection of lipids is generally determined by considerations such asliposome size and stability in the blood. A variety of methods areavailable for preparing liposomes as reviewed, for example, by (Coligan,J. E. et al, Current Protocols in Protein Science, 1799, John Wiley &Sons, Inc., New York) and see also U.S. Pat. Nos. 4,235,871, 4,501,728,4,837,028, and 5,017,369.

Liposomes containing the peptides or polypeptides of the invention canbe directed to the site of lymphoid cells where the liposomes thendeliver the selected immunogens directly to antigen presenting cells.Targeting can be achieved by incorporating additional molecules such asproteins or polysaccharides into the outer membranes of said structures,thus resulting in the delivery of the structures to particular areas ofthe body, or to particular cells within a given organ or tissue. Suchtargeting molecules may be a molecule that binds to receptor on antigenpresenting cells. For example an antibody that binds to CD80 could beused to direct liposomes to dendritic cells.

The immunogens of the present invention may also be administered assolid compositions. Conventional nontoxic solid carriers includingpharmaceutical grades of mannitol, lactose, starch, magnesium,cellulose, glucose, sucrose, sodium saccharin, and the like. Such solidcompositions will often be administered orally, whereby apharmaceutically acceptable nontoxic composition is formed byincorporating the peptides and polypeptides of the invention with any ofthe carriers listed above. Generally, such compositions will contain10-95% active ingredient, and more preferably 25-75% active ingredient.

Aerosol administration is also an alternative, requiring only that theimmunogens be properly dispersed within the aerosol propellant. Typicalpercentages of the peptides or polypeptides of the invention are0.01%-20% by weight, preferably 1%-10%. The use of a surfactant toproperly disperse the immunogen may be required. Representativesurfactants include the esters or partial esters of fatty acidscontaining from 6 to 22 carbon atoms, such as caproic, octanoic, lauric,palmitic, stearic, linoleic, linolenic, olesteric and oleic acids withan aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters,such as mixed or natural glycerides may be employed. The surfactant mayconstitute 0.1-20% by weight of the composition, preferably 0.25-5%.Typical propellants for such administration may include esters andsimilar chemicals but are by no means limited to these. A carrier, suchas lecithin for intranasal delivery, may also be included.

As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, basic salts of acid moietieswhich may be present on a peptide are prepared using a pharmaceuticallyacceptable base such as sodium hydroxide, potassium hydroxide, ammoniumhydroxide, calcium hydroxide, trimethylamine or the like.

A preferred embodiment of the pharmaceutical compositions comprise thepeptides as salts of acetic acid (acetates), ammonium or hydrochloricacid (chlorides).

In another embodiment, a pharmaceutical composition of the presentinvention may include sugars, sugar alcohols, amino acids such asglycine, arginine, glutaminic acid and others as framework former. Thesugars may be mono-, di- or trisaccharide. These sugars may be usedalone, as well as in combination with sugar alcohols. Examples of sugarsinclude glucose, mannose, galactose, fructose or sorbose asmonosaccharides, saccharose, lactose, maltose or trechalose asdisaccharides and raffinose as a trisaccharide. A sugar alcohol may be,for example, manniose, Preferred ingredients are saccharose, lactose,maltose, trehalose, mannitol and/or sorbitol, and more preferably,mannitol.

Pharmaceutical compositions of the present invention may includephysiological well tolerated excipients (see Handbook of PharmaceuticalExcipients, 5^(th) ed., edited by Raymond Rowe, Paul Shesky and SienOwen, Pharmaceutical Press (2006), such as antioxidants like ascorbicacid or glutathione, preserving agents such as phenole, m-cresole,methyl- or propylparabene, chlorobutanol, thiomersal orbenzalkoiumchloride, stabilizer, framework former such as saccharose,lactose, maltose, trehalose, mannitose, mannit and/or sorbit, mannitand/or lactose and solubilizer such as polyethyleneglycols (PEG), i.e.PEG 3000, 3350, 4000, or 6000, or cyclodextrines, i.e.hydroxypropyle-β-cyclodextrine, sulfobutylethyl-β-cyclodextrine orγ-cyclodextrine, or dextranes or poploxaolmers, i.e. poloxaomer 407,poloxamer 188, or Tween 20, Twee 80. In one embodiment, pharmaceuticalcompositions of the present invention include one or more well toleratedexcipients, selected from the group consisting of antioxidants,framework formers and stabilizers.

The peptides and polypeptides of the invention may also be deliveredwith an adjuvant. Adjuvants include, but are not limited to complete orincomplete Freund's adjuvant, Montanide ISA-51, Lymphocyte ActivationGene-3 (LAG-3), Toll like receptors (TLR), bacterial cell wall products,Lymphocyte Activation Gene-3 (LAG-3), Toll like receptors (TLR),bacterial cell wall products, aluminum phosphate, aluminum hydroxide,alum, and saponin. Adjuvant effects can also be obtained by injecting avariety of cytokines along with the immunogens of the invention. Thesecytokines include, but are not limited to IL-1, IL-2, IL-7, IL-12, andGM-CSF.

The peptides and polypeptides of the invention can also be added toprofessional antigen presenting cells such as dendritic cells that havebeen prepared ex vivo. For example, the dendritic cells could beprepared from CD34 positive stem cells from the bone marrow, or theycould be prepared from CD14 positive monocytes obtained from theperipheral blood. The dendritic cells are generated ex vivo usingcytokines such as GM-CSF, IL-3, IL4, TNF, and SCF. The cultured DC arethen pulsed with peptides at various concentrations using standardmethods that are well known in the art. The peptide-pulsed dendriticcells can then be administered intravenously, subcutaneously, orintradermally, and the immunization may also include cytokines such asIL-2 or IL-12.

The present invention is also directed to a vaccine in which animmunogen of the present invention is delivered or administered in theform of a polynucleotide encoding the a polypeptide or active fragmentas disclosed herein, whereby the peptide or polypeptide or activefragment is produced in vivo. The polynucleotide may be included in asuitable expression vector and combined with a pharmaceuticallyacceptable carrier. For example, the peptides or polypeptides could beexpressed in plasmid DNA and nonreplicative viral vectors such asvaccinia, fowlpox, Venezuelan equine encephalitis virus, adenovirus, orother RNA or DNA viruses. These examples are meant to be illustrativeonly and should not be viewed as self-limiting A wide variety of othervectors are available and are apparent to those skilled in the art fromthe description given herein. In this approach, a portion of thenucleotide sequence of the viral vector is engineered to express thepeptides or polypeptides of the invention. Vaccinia vectors and methodsuseful in immunization protocols are described in U.S. Pat. No.4,722,848, the disclosure of which is incorporated herein by referencein its entirety.

Regardless of the nature of the composition given, additionaltherapeutic agents may also accompany the immunogens of the presentinvention. Thus, for purposes of preventing or treating HBV infection,compositions containing the immunogens disclosed herein may, inaddition, contain other anti-viral pharmaceuticals. The use of suchcompositions with multiple active ingredients is left to the discretionof the clinician.

In addition, the immunogens of the present invention can be used tostimulate the production of antibodies for use in passive immunotherapy,for use as diagnostic reagents, and for use as reagents in otherprocesses such as affinity chromatography.

The present invention also relates to antibodies that react withimmunogens, such as a polypeptide comprising one or more of the epitopicpeptides of SEQ ID NO: 1-17 as disclosed herein. Active fragments ofsuch antibodies are also specifically contemplated.

Such antibodies, and active fragments of such antibodies, for example,and Fab structure, may react with, including where it is highlyselective or specific for, an immunogenic structure comprising 2, 3, 4or more of the epitopic peptides of the invention.

With the advent of methods of molecular biology and recombinanttechnology, it is now possible to produce antibody molecules byrecombinant means and thereby generate gene sequences that code forspecific amino acid sequences found in the polypeptide structure of theantibodies. Such antibodies can be produced by either cloning the genesequences encoding the polypeptide chains of said antibodies or bydirect synthesis of said polypeptide chains, with in vitro assembly ofthe synthesized chains to form active tetrameric (H.sub.2L.sub.2)structures with affinity for specific epitopes and antigenicdeterminants. This has permitted the ready production of antibodieshaving sequences characteristic of neutralizing antibodies fromdifferent species and sources.

Regardless of the source of the antibodies, or how they arerecombinantly constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, such as cows, goats and sheep, usinglarge cell cultures of laboratory or commercial size, in bioreactors orby direct chemical synthesis employing no living organisms at any stageof the process, all antibodies have a similar overall 3 dimensionalstructure. This structure is often given as H.sub.2L.sub.2 and refers tothe fact that antibodies commonly comprise 2 light (L) amino acid chainsand 2 heavy (H) amino acid chains. Both chains have regions capable ofinteracting with a structurally complementary antigenic target. Theregions interacting with the target are referred to as “variable” or “V”regions and are characterized by differences in amino acid sequence fromantibodies of different antigenic specificity.

The variable regions of either H or L chains contains the amino acidsequences capable of specifically binding to antigenic targets. Withinthese sequences are smaller sequences dubbed “hypervariable” because oftheir extreme variability between antibodies of differing specificity.Such hypervariable regions are also referred to as “complementaritydetermining regions” or “CDR” regions. These CDR regions account for thebasic specificity of the antibody for a particular antigenic determinantstructure.

The CDRs represent non-contiguous stretches of amino acids within thevariable regions but, regardless of species, the positional locations ofthese critical amino acid sequences within the variable heavy and lightchain regions have been found to have similar locations within the aminoacid sequences of the variable chains. The variable heavy and lightchains of all antibodies each have 3 CDR regions, each non-contiguouswith the others (termed L1, L2, L3, H1, H2, H3) for the respective light(L) and heavy (H) chains. The accepted CDR regions have been describedby Kabat et al, J. Biol. Chem. 252:6609-6616 (1777).

In all mammalian species, antibody polypeptides contain constant (i.e.,highly conserved) and variable regions, and, within the latter, thereare the CDRs and the so-called “framework regions” made up of amino acidsequences within the variable region of the heavy or light chain butoutside the CDRs.

The antibodies disclosed according to the invention may also be whollysynthetic, wherein the polypeptide chains of the antibodies aresynthesized and, possibly, optimized for binding to the polypeptidesdisclosed herein as being receptors. Such antibodies may be chimeric orhumanized antibodies and may be fully tetrameric in structure, or may bedimeric and comprise only a single heavy and a single light chain. Suchantibodies may also include fragments, such as Fab and F(ab.sub.2)′fragments, capable of reacting with and binding to any of thepolypeptides disclosed herein as being receptors.

A further embodiment of the present invention relates to a method forinducing a CTL response in a subject comprising administering tosubjects that express HLA A2 or B7 supertype antigens an effective(i.e., CTL-stimulating amount) of an immunogen of the invention thatdoes not comprise the entire protein expressing the epitopic peptidesdisclosed herein (i.e., one that comprises less than the entire proteinwhere the protein is a naturally occurring polypeptide) in an amountsufficient to induce a CTL response to HBV infected cells expressing atleast HLA-A2 or HLA-B7, as the case may be, thereby eliciting a cellularresponse against said HBV infected cells.

A still further embodiment of the present invention relates to a methodfor inducing a CTL response in a subject, wherein the immunogen is inthe form of a polynucleotide. In one non-limiting example, the methodcomprises administering to subjects that express HLA-A2 at least one CTLepitope, wherein said epitope or epitopes are selected from a groupcomprising the peptides disclosed according to the invention, and arecoded within a polynucleotide sequence that does not comprise the entireprotein coding region, in an amount sufficient to induce a CTL responseto HBV infected cells expressing HLA-A2.

While the below examples are provided to illustrate the invention, it isto be understood that these methods and examples in no way limit theinvention to the embodiments described herein and that other embodimentsand uses will no doubt suggest themselves to those skilled in the art.All publications, patents, and patent applications cited herein arehereby incorporated by reference, as are the references cited therein.It is also to be understood that throughout this disclosure where thesingular is used, the plural may be inferred and vice versa and use ofeither is not to be considered limiting. It should be borne in mind thatalthough these examples recite specific oligopeptide sequences of theinvention, as well as specific cell lines, the methodology disclosed inthe examples applies equally, with any obvious modifications, to use ofthe other oligopeptides and cell lines disclosed herein according to thepresent invention.

EXAMPLE

The HLA-A2 and A24 positive liver hepatocellular carcinoma cell lineHepG2 and its hepatitis B infected derivatives HepDE19 and HepG2.2.15were cultured in Dulbecco's Modified Eagle Medium/Ham's F-12 50/50 Mix(Mediatech Inc, Manassas Va.). 293-T cells were maintained inDulbecco's′ Modified Eagle Medium and T2 cells were maintained inRPMI-1640 (Mediatech Inc). All media was supplemented with 10% fetalbovine serum (Atlanta Biologicals, Flowery Branch, Ga.), L-glutamine(300 mg/mL), 1× non-essential amino acids, 0.5 mM sodium pyruvate, and1× penicillin/streptomycin (Mediatech Inc). Cells were maintained at 37°C. and 5% CO₂.

Cell lysates were prepared from HBV infected cells and MHC/peptidecomplexes were isolated by immunoaffinity chromatography using WICmolecule specific antibodies (Testa et al. 2012). The peptides purifiedfrom the MHC molecules were fractionated using C-18 reversed phase (RP)column (4.6 mm diameter×150 mm length) using an offline HPLC (Dionex,Sunnyvale, Calif.). The peptide containing fractions were collected anddried to 6 μL under vacuum for LC/MS/MS analysis.

Mass spectrometry experiments were carried out using LTQ (Thermo) andOrbitrap instruments interfaced with nano ultimate HPLC (Dionex).RP-HPLC purified peptide fractions were injected individually into theLC-MS/MS system to identify the sequences of the peptides. The peptideswere analyzed using a Data-Dependent method. The acquired spectra datawere searched against all influenza strains protein database usingProteome Discoverer (Thermo) to interpret data and derive peptidesequences.

Synthetic peptides were made and subjected to LC-MS/MS analysis underidentical experimental conditions as described above and their sequenceswere confirmed based on their MS/MS data. Candidate peptide sequenceswere confirmed by comparison of their MS/MS spectra with that of theirsynthetic analogs.

In vitro peptide specific CTLs were generated using heparinized bloodfrom healthy HLA-A2+ donors purchased from Research Blood Components,LLC (Brighton, Mass.). Peripheral blood mononuclear cells (PBMC) werepurified using differential centrifugation following standard methods.PBMC were used to generate peptide specific CTL as described previously(Testa et al. 2012).

In vivo peptide specific CTLs were generated using HLA-A2 and HLA-A24transgenic mice. Mice were injected with PBS alone or bug of syntheticpeptides in PBS or a 50:50 emulsion with Montanide ISV 51 (Seppic Inc,Fairfield, N.J.). Mice received a total of three injections, at 10 daysintervals. A week after the third injection, mice were euthanized andthe spleens harvested for use in T cell functional assays.

Antigen specific CTL activation was assessed by interferon-γ (IFN-γ)release as a measure using an ELISPOT assay (BD-Pharmingen, San Jose,Calif.) and a customized MILLIPLEX magnetic bead assay and CD107a(degranulation marker) expression by flow cytometry.

Seventeen epitopes including HLA-A2 and A24 specific motifs wereidentified (Table 1). All the peptide sequences were present in multiplegenotype of HBV family. Eight HLA-A2 and A24 specific epitopes (SEQ ID:1-8) were selected for CTL characterization. Synthetic peptides wereused for CTL analysis.

TABLE 1 List of identified HBV MHC peptides, their se-quences, corresponding proteins and accession ID's Seq Acces- ID PeptideParent Protein sion ID  1 FLGGPPVCL Surface (S) Q0EED2  2 ILRSFIPLLSurface (S) Q6WYY8  3 FLKQQYMNL Polymerase (P) I0DE20  4 FLSKQYMDLPolymerase (P) L7QBE1  5 TVSTKLCKI Polymerase (P) Q8B4E6  6 GGPNLDNILLarge E Q8QSF2  7 LTTVPAASLLA Large E Q9YKJ7  8 LTFGRETVLENPrecore/Core (C) Q6UFV9  9 IYDHQHGTL Polymerase (P) C9ED71 10TVLENLVSLGV precore/core protein gi34419971 [Hepatitis B virus] 11QAMQWNSLAF large S protein  gi222824657 [Hepatitis B  virus] 12 IEANKVGVpreS1 protein [Hepa-  gi164509420 titis B virus] 13 AQGTLTSVPVlarge S protein  gi283971254 [Hepatitis B virus] 14 VLLDCQGMLLhepatitis surface gi94466780 antigen [Hepatitis  B virus] 15 MAARLRCQLX protein [Hepa-  gi260184403 titis B virus] 16 KVCRRIVGLLGFApolymerase [Hepa-  gi225675980 titis B virus] 17 NTNMGLKILQLLWpre-C/C protein  gi164416566 [Hepatitis B virus]

Identification of MHC Class I Presented Epitopes from Hepatitis B VirusInfected Cells by Nano LC/MS/MS Methods

MHC class I associated peptides isolated from HBV infected cells weresubjected to LC/MS/MS analysis to identify the peptides and theircorresponding proteins. Employing this strategy, we identified seventeenMHC associated peptides (Seq ID: 1-17). Prior to CTL characterizationexperiments, we confirmed the authenticity of the peptides and selectedfive HLA-A2 and A24 specific peptides (Seq ID: 1-8) using theirsynthetic peptide analogs.

Epitopes Identified by Immunoproteomics Analysis Activate HBV SpecificCTLs In Vitro

To verify the presentation of these epitopes by infected cells, CTLsspecific for the peptides were generated using PBMCs from healthyHLA-A2+ donors and synthetic peptides corresponding to the identifiedepitopes. In ELISpot assays, CTL functionality was measured by detectionof antigen specific IFNγ secretion. As illustrated in FIG. 1, epitopespecific CD8⁺ T cells were activated (as measured by IFN-gamma ELISpot)when cultured with T2s pulsed with peptides (FIG. 1A) and with HepDE19or 293-T/A2-AdHBV cell lines (FIG. 1B). Importantly, these responseswere specific as no CD8⁺ T cell activation was observed when the cellswere cultured with normal liver, uninfected HepG2, or uninfected 293T/A2cells (FIG. 1B)

Epitopes Identified by Immunoproteomics Analysis Activate HBV SpecificCTLs In Vivo

After establishing that epitopes could specifically induce CD8⁺ T cellactivation in vitro, we next determined if the experimentally identifiedpeptides could also stimulate CD8⁺ T cells in vivo. Because a subset ofour peptides (P4-P8) had the motif to bind both HLA-A2 and HLA-A24molecules, we assessed CD8⁺ T cell activation of these peptides in bothcontexts. Synthetic versions of peptides were injected into HLA-A2⁺ orHLA-A24⁺ transgenic mice with or without Montanide ISV-51, three timesin total. One week after the third injection, splenocytes were harvestedand cultured with HepG2 cells pulsed individually with peptides in anIFN-gamma ELISPot assay. As shown in FIG. 2A, CD8⁺ T cells generated invivo specifically recognized peptide loaded HepG2 cells as well as HBVinfected HepDE19 cells. In addition, CD8⁺ T cells generated in vivo alsoupregulated a classical marker of degranulation (CD107a) (Betts et al.2003, Mittendorf et al. 2005) after stimulation with both peptide pulsedHepG2s and the HBV infected cell line HepDE19 (FIG. 2B). Interestingly,the peptides induced IFN-gamma secretion and CD107a upregulationindependent of the HLA molecule tested which indicates that peptides arecapable of binding both HLA-A2 and HLA-A24 molecules.

Because degranulation is associated with delivery of perforin andgranzyme to target cells, we next checked the levels of granzyme B beingsecreted by the peptide activated CD8⁺ T cells. Supernatants werecollected from the stimulated cells and the levels of granzyme B weredetected using Milliplex magnetic bead technology (Parmigiani et al.2013; Nieminen et al. 2013). CD8⁺ T cells from both HLA-A2 and HLA-A24mice secreted high levels of granzyme-B in response to peptidestimulation and HepDE19 stimulation than their naïve counterpartsindicating the activation of a specific cytotoxic response (FIG. 3).

SEQUENCE LISTING Seq  ID Acces- No Peptide Protein sion ID Seq  FLGGPPVCL Surface (S) Q0EED2 ID No 1 Seq  ILRSFIPLL Surface (S) Q6WYY8ID No 2 Seq  FLKQQYMNL Polymerase (P) I0DE20 ID No 3 Seq  FLSKQYMDLPolymerase (P) L7QBE1 ID No 4 Seq  TVSTKLCKI Polymerase (P) Q8B4E6 IDNo 5 Seq  GGPNLDNIL Large E Q8QSF2 ID No 6 Seq  LTTVPAASLLA Large EQ9YKJ7 ID No 7 Seq  LTFGRETVLEN Precore/Core  Q6UFV9 ID (C) No 8 Seq IYDHQHGTL Polymerase (P) C9ED71 ID No 9 Seq   TVLENLVSLGV precore/coregi34419971 ID protein [Hepa-  No 10 titis B virus] Seq   QAMQWNSLAFlarge S protein  gi222824657 ID [Hepatitis B No 11 virus] Seq   IEANKVGVpreS1 protein  gi164509420 ID [Hepatitis B  No 12 virus] Seq  AQGTLTSVPV large S protein  gi283971254 ID [Hepatitis B  No 13 virus]Seq   VLLDCQGMLL hepatitis sur-  gi94466780 ID face antigen No 14[Hepatitis  B virus] Seq   MAARLRCQL X protein  gi260184403 ID[Hepatitis B  No 15 virus] Seq   KVCRRIVGLLGFA polymerase  gi225675980ID [Hepatitis B  No 16 virus] Seq   NTNMGLKILQLLW pre-C/C proteingi164416566 ID [Hepatitis B  No 17 virus]

1. A pharmaceutical composition comprising a polypeptide, oligopeptideor peptide consisting of 8 to about 30 amino acid residues andcomprising a sequence that is selected from the group consisting of: (i)SEQ ID NO: 1, 3, and 5 to 17; and (ii) an amino acid sequence thatdiffers from SEQ ID NO: 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,or 17 by no more than one amino acid unit; wherein said polypeptide,oligopeptide or peptide binds to one or more class I MHC alleles or canbe processed to bind to one or more class I MHC alleles and activate a Tlymphocyte.
 2. The pharmaceutical composition of claim 1 wherein saidpolypeptide, oligopeptide or peptide comprises at least two epitopicpeptides.
 3. The pharmaceutical composition of claim 1 wherein saidpolypeptide, oligopeptide or peptide comprises at least three epitopicpeptides.
 4. The pharmaceutical composition of claim 1 wherein saidpolypeptide, oligopeptide or peptide comprises at least four epitopicpeptides.
 5. A polynucleotide selected from the group consisting of: (a)a polynucleotide that encodes an oligopeptide or peptide of claim 1, and(b) the full complement of (a).
 6. The polynucleotide of claim 5,wherein the polynucleotide of (a) is DNA.
 7. The polynucleotide of claim5, wherein the polynucleotide of (a) is RNA.
 8. A method for vaccinatingand treating a subject for HBV infection, said infected cells expressingany class I MHC molecule, comprising administering to said subject acomposition that binds to class I MHC molecules or can be processed tobind to class I MHC molecules comprising: at least one polypeptidecomprising an epitopic peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, 3, and 5 to 17 in anamount sufficient to induce a CTL response to said infected cells; or atleast one polypeptide comprising an epitopic peptide having no more thanone amino acid difference from an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, 3, and 5 to 17 in an amount sufficientto induce a CTL response to said infected cells.
 9. A method forvaccinating and treating a subject with HBV infection, said infectedcells expressing any class I MHC molecule, said method comprisingadministering to said subject a composition that binds to class I MHCmolecules or can be processed to bind to class I MHC moleculescomprising: a polynucleotide comprising a nucleic acid sequenceencoding: at least one polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, 3, and 5 to 17 in anamount sufficient to induce a CTL response to said infected cells; or atleast one polypeptide comprising an epitopic peptide comprising no morethan one amino acid difference from an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1, 3, and 5 to 17 in an amountsufficient to induce a CTL response to said infected cells.
 14. A methodfor generating an immune response ex vivo using T cells from a subjectinfected with HBV, said method comprising: stimulating the production ofCTL response for use in passive immunotherapy, wherein said T cellsreact with at least one polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, 3, and 5 to 17; orat least one polypeptide comprising no more than one amino aciddifference from an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, 3, and 5 to
 17. 15. The method of claim 14,wherein said method is a T cell adoptive therapy generated fromautologous or HLA matched subjects.
 16. A method for assessing ordiagnosing an immune response in a subject infected with HBV orvaccinated for HBV and related viruses said method comprising:stimulating the production of CTL response, wherein said T cells reactwith at least one polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 3, and 5 to 17; or at leastone polypeptide comprising no more than one amino acid difference froman amino acid sequence selected from the group consisting of SEQ ID NO:1, 3, and 5 to
 17. 17. A method for vaccinating humans against HBVinfection using the pharmaceutical composition of claim 1.